Inhibition of protein degradation is an emerging anti-cancer strategy. The proteasome inhibitor Bortezomib has been approved by FDA for treatment of multiple myeloma, and is being trialed in numerous cancers. However, the underlying molecular mechanisms remain elusive. Moreover, it has been unclear whether certain molecular signatures can be used to predict the outcome of proteasome inhibitor-based therapy. We recently reported that proteasome inhibitors can induce an intracellular aggregation and activation of caspase-8 and subsequent apoptosis. This caspase-8 activation is mediated by its association with a ubiquitin-binding protein SQSTM1/p62 and an autophagy-related protein microtubule-associated protein light chain 3 (LC3). These findings prompt us to hypothesize that there exists a previously uncharacterized cell death mechanism that involves protein aggregate formation and intracellular activation of caspase-8. Along this direction, our additional preliminary results show that p62 itself undergoes ubiquitination. This novel modification of p62 may play a critical function in mediating aggregate formation and apoptosis. We also reported that an endogenous serine/cysteine protease inhibitor SerpinB3 (also termed squamous cell carcinoma antigen 1, SCCA1) may function as a molecular signature for predicting efficacy with proteotoxicity-based anti-cancer therapy. SCCA1 is an intracellular serpin that inhibits lysosomal proteases. SCCA1 is believed to limit cellular damage resulting from unscheduled activation of lysosomal protease that is detrimental to the cell, hence may contribute to tumorigenesis and chemo-resistance. Studies including those recently from my laboratory have demonstrated that elevated SCCA1 expression is associated with poorer prognosis in numerous advanced human cancers such as squamous cell carcinomas of lung, head and neck, and esophagus, as well as hepatocellular carcinoma and breast carcinoma. Indeed, at the molecular level, we found that SCCA1 protects cells from lysosomal injury induced by DNA alkylating damage and oxidative stress. On the other hand, we also found that SCCA1 promotes apoptosis in response to proteotoxic stress. Therefore, SCCA1 on one hand may confer resistance to chemotherapy by protecting cells against lysosomal injury, on the other hand, it may sensitize cancer cells to proteotoxicity. This proposal is designed to understand the molecular mechanisms underlying the anti-tumor effect of proteasome inhibitors, and to determine whether certain molecular changes in cancer cells such as elevated expression of LC3 or SCCA1 can confer tumor cell sensitive to proteotoxic agents in vivo. We propose three molecularly and clinically related Specific Aims: 1) Characterize the activation of caspase-8 upon the inhibition of proteasome degradation. 2) Study the mechanisms through which p62 regulates aggregate formation and caspase-8 activation. 3) Examine the hypothesis that certain molecules such as LC3 and SCCA1 can sensitize tumors to proteotoxic agents in vivo. Accomplishing this project will have a general impact on the understanding of the molecular basis for proteotoxicity-based anti-cancer therapy, as well as on the physiological relevance of protein aggregates in many pathological conditions. At the clinical level, it may establish LC3 or SCCA1 as a molecular signature for suggesting treatment with proteotoxic agents.